GRG36: Novel EPSP Synthase Gene Conferring Herbicide Resistance

ABSTRACT

Compositions and methods for conferring herbicide resistance to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include nucleic acid molecules encoding herbicide resistance or tolerance polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. The nucleotide sequences of the invention can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, the present invention provides for isolated nucleic acid molecules comprising the nucleotide sequence set forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22, the herbicide resistance nucleotide sequence deposited in a bacterial host as Accession Nos. NRRL B-30932, B-30933, B-30934, B-30945, B-30946, B-30947, or B-30948, as well as variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.11/769,327, filed Jun. 27, 2007, which claims the benefit of U.S.Provisional Application Ser. Nos. 60/816,676, filed Jun. 27, 2006;60/819,122, filed Jul. 7, 2006; and, 60/819,119, filed Jul. 7, 2006, thecontents of which are herein incorporated by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“APA046US01DSEQLIST.txt”, created on Apr. 26, 2011, and having a size of150 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention provides novel genes encoding5-enolpyruvylshikimate-3-phosphate (EPSP) synthase that provideherbicide resistance. These genes are useful in plant biology, cropbreeding, and plant cell culture.

BACKGROUND OF THE INVENTION

N-phosphonomethylglycine, commonly referred to as glyphosate, is animportant agronomic chemical. Glyphosate inhibits the enzyme thatconverts phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to5-enolpyruvyl-3-phosphoshikimic acid. Inhibition of this enzyme(5-enolpyruvylshikimate-3-phosphate synthase; referred to herein as“EPSP synthase”) kills plant cells by shutting down the shikimatepathway, thereby inhibiting aromatic acid biosynthesis.

Since glyphosate-class herbicides inhibit aromatic amino acidbiosynthesis, they not only kill plant cells, but are also toxic tobacterial cells. Glyphosate inhibits many bacterial EPSP synthases, andthus is toxic to these bacteria. However, certain bacterial EPSPsynthases have a high tolerance to glyphosate.

Plant cells resistant to glyphosate toxicity can be produced bytransforming plant cells to express glyphosate-resistant bacterial EPSPsynthases. Notably, the bacterial gene from Agrobacterium tumefaciensstrain CP4 has been used to confer herbicide resistance on plant cellsfollowing expression in plants. A mutated EPSP synthase from Salmonellatyphimurium strain CT7 confers glyphosate resistance in bacterial cells,and confers glyphosate resistance on plant cells (U.S. Pat. Nos.4,535,060; 4,769,061; and 5,094,945).

U.S. Pat. No. 6,040,497 reports mutant maize EPSP synthase enzymeshaving substitutions of threonine to isoleucine at position 102 andproline to serine at position 106 (the “TIPS” mutation). Suchalterations confer glyphosate resistance upon the maize enzyme. Amutated EPSP synthase from Salmonella typhimurium strain CT7 confersglyphosate resistance in bacterial cells, and is reported to conferglyphosate resistance upon plant cells (U.S. Pat. Nos. 4,535,060;4,769,061; and 5,094,945). He et al. ((2001) Biochim et Biophysica Acta1568:1-6) have developed EPSP synthases with increased glyphosatetolerance by mutagenesis and recombination between the E. coli andSalmonella typhimurium EPSP synthase genes, and suggest that mutationsat position 42 (T42M) and position 230 (Q230K) are likely responsiblefor the observed resistance.

Subsequent work (He et al. (2003) Biosci. Biotech. Biochem.67:1405-1409) shows that the T42M mutation (threonine to methionine) issufficient to improve tolerance of both the E. coli and Salmonellatyphimurium enzymes. These enzymes contain amino acid substitutions intheir active sites that prevent the binding of glyphosate withoutaffecting binding by PEP or S3P. Mutations that occur in the hingeregion between the two globular domains of EPSP synthase have been shownto alter the binding affinity of glyphosate but not PEP (He et al.,2003, supra). Therefore, such enzymes have high catalytic activity, evenin the presence of glyphosate.

Due to the many advantages herbicide resistance plants provide, methodsfor identifying herbicide resistance genes with glyphosate resistanceactivity are desirable.

SUMMARY OF INVENTION

Compositions and methods for conferring herbicide resistance ortolerance to bacteria, plants, plant cells, tissues and seeds areprovided. Compositions include nucleic acid molecules encoding herbicideresistance or tolerance polypeptides, vectors comprising those nucleicacid molecules, and host cells comprising the vectors. Compositions alsoinclude antibodies to the herbicide resistance or tolerancepolypeptides. As noted the nucleotide sequences of the invention can beused in DNA constructs or expression cassettes for transformation andexpression in organisms, including microorganisms and plants.Compositions also comprise transformed bacteria, plants, plant cells,tissues, and seeds. In addition, methods are provided for producing thepolypeptides encoded by the synthetic nucleotides of the invention.

In particular, isolated nucleic acid molecules and variants thereofencoding herbicide resistance- or tolerance polypeptides are provided.Additionally, amino acid sequences and variants thereof encoded by thepolynucleotides that confer herbicide resistance or tolerance areencompassed. In particular, the present invention provides for isolatednucleic acid molecules comprising the nucleotide sequence set forth inSEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, anucleotide sequence encoding the amino acid sequence shown in SEQ IDNO:2, 5, 8, 11, 14, 16, 19, or 22, the herbicide resistance nucleotidesequence deposited in a bacterial host as Accession Nos. NRRL B-30932,B-30933, B-30934, B-30945, B-30946, B-30947, or B-30948, as well asvariants and fragments thereof. Nucleotide sequences that arecomplementary to a nucleotide sequence of the invention, or thathybridize to a sequence of the invention or a complement of a sequenceof the invention are also encompassed.

DESCRIPTION OF FIGURES

FIG. 1 shows an alignment of the amino acid sequence of GRG33 (SEQ IDNO:2) and GRG35 (SEQ ID NO:5) with EPSP synthase sequences fromStreptomyces cooelicolor (SEQ ID NO:24), Streptomyces avermitilis (SEQID NO:25), Zea mays (SEQ ID NO:38), and E. coli (SEQ ID NO:37). Thealignment shows the most highly conserved amino acid residueshighlighted in black and highly conserved amino acid residueshighlighted in gray.

FIG. 2 shows an alignment of the amino acid sequence of GRG36 (SEQ IDNO:8) with EPSP synthase sequences from Bacillus halodurans (SEQ IDNO:26), Bacillus claussi (SEQ ID NO:27), Zea mays (SEQ ID NO:38), and E.coli (SEQ ID NO:37). The alignment shows the most highly conserved aminoacid residues highlighted in black and highly conserved amino acidresidues highlighted in gray.

FIG. 3 shows an alignment of GRG38 (SEQ ID NO:16) and GRG50 (SEQ IDNO:22) with other EPSP synthase enzymes, including GRG8 (SEQ ID NO:29),GRG12 (SEQ ID NO:30), GRG15 (SEQ ID NO:31), GRG5 (SEQ ID NO:32), GRG6(SEQ ID NO:33), GRG7 (SEQ ID NO:34), GRG9 (SEQ ID NO:35), GRG1 (SEQ IDNO:41), the EPSP synthase described in International Patent ApplicationNo. WO2005014820 (SEQ ID NO:36), and EPSP synthase enzymes from E. coli(SEQ ID NO:37), Zea mays (SEQ ID NO:38), Agrobacterium tumefaciens (SEQID NO:39), and Bacillus subtilis (SEQ ID NO:40). The alignment shows themost highly conserved amino acid residues highlighted in black, andhighly conserved amino acid residues highlighted in gray.

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating herbicide resistance in organisms, particularly in plants orplant cells. The methods involve transforming organisms with anucleotide sequence encoding a glyphosate resistance gene of theinvention. In particular, a nucleotide sequence of the invention isuseful for preparing plants that show increased tolerance to theherbicide glyphosate. Thus, transformed bacteria, plants, plant cells,plant tissues and seeds are provided.

Compositions include nucleic acids and proteins relating to herbicidetolerance in microorganisms and plants as well as transformed bacteria,plants, plant tissues and seeds. More particularly, nucleotide sequencesof the glyphosate resistance genes (grg33, syngrg33, grg35, syngrg35,grg36, syngrg36, grg37, syngrg37, grg38, syngrg38, grg39, syngrg39,grg50, syngrg50) and the amino acid sequences of the proteins encodedthereby are disclosed. The sequences find use in the construction ofexpression vectors for subsequent transformation into plants ofinterest, as probes for the isolation of other glyphosate resistancegenes, as selectable markers, and the like. Thus, by “glyphosateresistance gene of the invention” is intended the nucleotide sequenceset forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21,or 23, and fragments and variants thereof that encode a glyphosateresistance or tolerance polypeptide. Likewise, a “glyphosate resistancepolypeptide of the invention” is a polypeptide having the amino acidsequence set forth in SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22, andfragments and variants thereof that confer glyphosate resistance ortolerance to a host cell.

Plasmids containing the herbicide resistance nucleotide sequences of theinvention were deposited in the permanent collection of the AgriculturalResearch Service Culture Collection, Northern Regional ResearchLaboratory (NRRL), 1815 North University Street, Peoria, Ill. 61604,United States of America, on Jun. 9, 2006, and assigned Accession Nos.NRRL B-30932 (for grg33), NRRL B-30933 (for grg35), and NRRL B-30934(for grg36); and on Jun. 26, 2006, and assigned Accession Nos. NRRLB-30945 (for grg37), NRRL B-30946 (for grg38), NRRL B-30947 (for grg39),and NRRL B-30948 (for grg50). This deposit will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. Accessto these deposits will be available during the pendency of theapplication to the Commissioner of Patents and Trademarks and personsdetermined by the Commissioner to be entitled thereto upon request. Uponallowance of any claims in the application, the Applicants will makeavailable to the public, pursuant to 37 C.F.R. §1.808, sample(s) of thedeposit with the ATCC. This deposit was made merely as a convenience forthose of skill in the art and is not an admission that a deposit isrequired under 35 U.S.C. §112.

By “glyphosate” is intended any herbicidal form ofN-phosphonomethylglycine (including any salt thereof) and other formsthat result in the production of the glyphosate anion in planta. An“herbicide resistance protein” or a protein resulting from expression ofan “herbicide resistance-encoding nucleic acid molecule” includesproteins that confer upon a cell the ability to tolerate a higherconcentration of an herbicide than cells that do not express theprotein, or to tolerate a certain concentration of an herbicide for alonger period of time than cells that do not express the protein. A“glyphosate resistance protein” includes a protein that confers upon acell the ability to tolerate a higher concentration of glyphosate thancells that do not express the protein, or to tolerate a certainconcentration of glyphosate for a longer period of time than cells thatdo not express the protein. By “tolerate” or “tolerance” is intendedeither to survive, or to carry out essential cellular functions such asprotein synthesis and respiration in a manner that is not readilydiscernable from untreated cells.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding herbicideresistance proteins and polypeptides or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify herbicide resistance-encoding nucleicacids. As used herein, the term “nucleic acid molecule” is intended toinclude DNA molecules (e.g., cDNA, recombinant DNA, or genomic DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecules can be single-stranded ordouble-stranded, but preferably are double-stranded DNA.

Nucleotide sequences encoding the proteins of the present inventioninclude the sequences set forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12,13, 15, 17, 18, 20, 21, or 23, the herbicide resistance nucleotidesequence deposited in a bacterial host as Accession Nos. NRRL B-30932,B-30933, B-30934, B-30945, B-30946, B-30947, or B-30948, and variants,fragments, and complements thereof. By “complement” is intended anucleotide sequence that is sufficiently complementary to a givennucleotide sequence such that it can hybridize to the given nucleotidesequence to thereby form a stable duplex. In some embodiments, thecomplement hybridizes across the full length of the sequence of theinvention. In another embodiment, the complement hybridizes across atleast about 50% of the sequence of the invention, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% of a sequence of the invention.The corresponding amino acid sequences for the herbicide resistanceproteins encoded by these nucleotide sequences are set forth in SEQ IDNO:2, 5, 8, 11, 14, 16, 19, or 22. The invention also encompassesnucleic acid molecules comprising nucleotide sequences encodingpartial-length herbicide resistance proteins, and complements thereof.

In some embodiments, the polynucleotides of the present invention encodepolypeptides that are Class III EPSP synthase enzymes. For the purposesof the present invention, a “Class III EPSP synthase enzyme” is anherbicide tolerant or herbicide resistant polypeptide containing one ormore of the amino acid sequence domains described in U.S. patentapplication Ser. No. 11/400,598, which is herein incorporated byreference in its entirety.

An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolatedglyphosate resistance-encoding nucleic acid molecule can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequence that naturally flanks the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. An herbicideresistance protein that is substantially free of cellular materialincludes preparations of protein having less than about 30%, 20%, 10%,or 5% (by dry weight) of non-herbicide resistance protein (also referredto herein as a “contaminating protein”).

Nucleic acid molecules that are fragments of these herbicideresistance-encoding nucleotide sequences are also encompassed by thepresent invention. By “fragment” is intended a portion of a nucleotidesequence encoding an herbicide resistance protein. A fragment of anucleotide sequence may encode a biologically active portion of anherbicide resistance protein, or it may be a fragment that can be usedas a hybridization probe or PCR primer using methods disclosed below.Nucleic acid molecules that are fragments of an herbicide resistancenucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 contiguousnucleotides, or up to the number of nucleotides present in a full-lengthherbicide resistance-encoding nucleotide sequence disclosed herein (forexample, 1329 nucleotides for SEQ ID NO:1; 1353 nucleotides for SEQ IDNO:4; 1344 nucleotides for SEQ ID NO:7, etc) depending upon the intendeduse. By “contiguous” nucleotides is intended nucleotide residues thatare immediately adjacent to one another.

Fragments of the nucleotide sequences of the present invention generallywill encode protein fragments that retain the biological activity of thefull-length glyphosate resistance protein; i.e., herbicide-resistanceactivity. By “retains herbicide resistance activity” is intended thatthe fragment will have at least about 30%, at least about 50%, at leastabout 70%, or at least about 80% of the herbicide resistance activity ofthe full-length glyphosate resistance protein disclosed herein as SEQ IDNO:2, 5, 8, 11, 14, 16, 19, or 22. Methods for measuring herbicideresistance activity are well known in the art. See, for example, U.S.Pat. Nos. 4,535,060, and 5,188,642, each of which are hereinincorporated by reference in their entirety.

A fragment of an herbicide resistance-encoding nucleotide sequence thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400 contiguous amino acids, or up to the total number of aminoacids present in a full-length herbicide resistance protein of theinvention (for example, 442 amino acids for SEQ ID NO:2; 450 for SEQ IDNO:5; 447 amino acids for SEQ ID NO:8, etc).

Preferred herbicide resistance proteins of the present invention areencoded by a nucleotide sequence sufficiently identical to thenucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17,18, 20, 21, or 23. The term “sufficiently identical” is intended anamino acid or nucleotide sequence that has at least about 60% or 65%sequence identity, about 70% or 75% sequence identity, about 80% or 85%sequence identity, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% sequence identity compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous toglyphosate-resistant nucleic acid molecules of the invention. BLASTprotein searches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to herbicideresistance protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. See www.ncbi.nlm.nih.gov. Anothernon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the ClustalW algorithm (Higgins et al. (1994)Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and alignsthe entirety of the amino acid or DNA sequence, and thus can providedata about the sequence conservation of the entire amino acid sequence.The ClustalW algorithm is used in several commercially availableDNA/amino acid analysis software packages, such as the ALIGNX module ofthe Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.).After alignment of amino acid sequences with ClustalW, the percent aminoacid identity can be assessed. A non-limiting example of a softwareprogram useful for analysis of ClustalW alignments is GENEDOC™. GENEDOC™(Karl Nicholas) allows assessment of amino acid (or DNA) similarity andidentity between multiple proteins. Another non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0), which is part ofthe GCG sequence alignment software package (available from Accelrys,Inc., 9865 Scranton Rd., San Diego, Calif., USA). When utilizing theALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) supra, will be used to determine sequenceidentity or similarity using the following parameters: % identity and %similarity for a nucleotide sequence using GAP Weight of 50 and LengthWeight of 3, and the nwsgapdna.cmp scoring matrix; % identity or %similarity for an amino acid sequence using GAP weight of 8 and lengthweight of 2, and the BLOSUM62 scoring program. Equivalent programs mayalso be used. By “equivalent program” is intended any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

The invention also encompasses variant nucleic acid molecules.“Variants” of the herbicide resistance-encoding nucleotide sequencesinclude those sequences that encode an herbicide resistance proteindisclosed herein but that differ conservatively because of thedegeneracy of the genetic code, as well as those that are sufficientlyidentical as discussed above. Naturally occurring allelic variants canbe identified with the use of well-known molecular biology techniques,such as polymerase chain reaction (PCR) and hybridization techniques asoutlined below. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences that have been generated, for example, byusing site-directed mutagenesis but which still encode the herbicideresistance proteins disclosed in the present invention as discussedbelow. Variant proteins encompassed by the present invention arebiologically active, that is they retain the desired biological activityof the native protein, that is, herbicide resistance activity. By“retains herbicide resistance activity” is intended that the variantwill have at least about 30%, at least about 50%, at least about 70%, orat least about 80% of the herbicide resistance activity of the nativeprotein. Methods for measuring herbicide resistance activity are wellknown in the art. See, for example, U.S. Pat. Nos. 4,535,060, and5,188,642, each of which are herein incorporated by reference in theirentirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation into the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedherbicide resistance protein, without altering the biological activityof the protein. Thus, variant isolated nucleic acid molecules can becreated by introducing one or more nucleotide substitutions, additions,or deletions into the corresponding nucleotide sequence disclosedherein, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Such variant nucleotide sequences are alsoencompassed by the present invention.

For example, conservative amino acid substitutions may be made at one ormore predicted, preferably nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe wild-type sequence of an herbicide resistance protein withoutaltering the biological activity, whereas an “essential” amino acidresidue is required for biological activity. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Amino acid substitutions may bemade in nonconserved regions that retain function. In general, suchsubstitutions would not be made for conserved amino acid residues, orfor amino acid residues residing within a conserved motif, where suchresidues are essential for protein activity. However, one of skill inthe art would understand that functional variants may have minorconserved or nonconserved alterations in the conserved residues.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in the alignment of FIG. 1, 2, or 3.Examples of residues that are conserved but that may allow conservativeamino acid substitutions and still retain activity include, for example,residues that have only conservative substitutions between all proteinscontained in the alignment of FIG. 1, 2, or 3.

Lys-22, Arg-124, Asp-313, Arg-344, Arg-386, and Lys-411, are conservedresidues of the EPSP synthase from E. coli (Schönbrunn et al. (2001)Proc. Natl. Acad. Sci. USA 98:1376-1380). Conserved residues importantfor EPSP synthase activity also include Arg-100, Asp-242, and Asp-384(Selvapandiyan et al. (1995) FEBS Letters 374:253-256). Arg-27 binds toS3P (Shuttleworth et al. (1999) Biochemistry 38:296-302).

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer herbicide resistance activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Using methods such as PCR, hybridization, and the like, correspondingherbicide resistance sequences can be identified, such sequences havingsubstantial identity to the sequences of the invention. See, forexample, Sambrook J., and Russell, D. W. (2001) Molecular Cloning: ALaboratory Manual. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methodsand Applications (Academic Press, NY).

In a hybridization method, all or part of the herbicide resistancenucleotide sequence can be used to screen cDNA or genomic libraries.Methods for construction of such cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook and Russell,2001, supra. The so-called hybridization probes may be genomic DNAfragments, cDNA fragments, RNA fragments, or other oligonucleotides, andmay be labeled with a detectable group such as ³²P, or any otherdetectable marker, such as other radioisotopes, a fluorescent compound,an enzyme, or an enzyme co-factor. Probes for hybridization can be madeby labeling synthetic oligonucleotides based on the known herbicideresistance-encoding nucleotide sequences disclosed herein. Degenerateprimers designed on the basis of conserved nucleotides or amino acidresidues in the nucleotide sequences or encoded amino acid sequences canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, at least about 50, 75, 100, 125,150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1200,1300 consecutive nucleotides of an herbicide resistance-encodingnucleotide sequence of the invention or a fragment or variant thereof.Methods for the preparation of probes for hybridization are generallyknown in the art and are disclosed in Sambrook and Russell, 2001, supraand Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),both of which are herein incorporated by reference.

For example, an entire herbicide resistance sequence disclosed herein,or one or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding herbicide resistance sequencesand messenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and are atleast about 10 nucleotides in length, or at least about 20 nucleotidesin length. Such probes may be used to amplify corresponding herbicideresistance sequences from a chosen organism by PCR. This technique maybe used to isolate additional coding sequences from a desired organismor as a diagnostic assay to determine the presence of coding sequencesin an organism. Hybridization techniques include hybridization screeningof plated DNA libraries (either plaques or colonies; see, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.Unless otherwise specified, hybridization conditions are under highstringency.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Herbicide resistance proteins are also encompassed within the presentinvention. By “herbicide resistance protein” is intended a proteinhaving the amino acid sequence set forth in SEQ ID NO:2, 5, 8, 11, 14,16, 19, or 22. Fragments, biologically active portions, and variantsthereof are also provided, and may be used to practice the methods ofthe present invention.

“Fragments” or “biologically active portions” include polypeptidefragments comprising a portion of an amino acid sequence encoding anherbicide resistance protein as set forth SEQ ID NO:2, 5, 8, 11, 14, 16,19, or 22, and that retains herbicide resistance activity. Abiologically active portion of an herbicide resistance protein can be apolypeptide that is, for example, 10, 25, 50, 100 or more amino acids inlength. Such biologically active portions can be prepared by recombinanttechniques and evaluated for herbicide resistance activity. Methods formeasuring herbicide resistance activity are well known in the art. See,for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which areherein incorporated by reference in their entirety. As used here, afragment comprises at least 8 contiguous amino acids of SEQ ID NO:2, 5,8, 11, 14, 16, 19, or 22. The invention encompasses other fragments,however, such as any fragment in the protein greater than about 10, 20,30, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, 80%, 85%, or90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22.Variants also include polypeptides encoded by a nucleic acid moleculethat hybridizes to the nucleic acid molecule of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, or a complement thereof, understringent conditions. Variants include polypeptides that differ in aminoacid sequence due to mutagenesis. Variant proteins encompassed by thepresent invention are biologically active, that is they continue topossess the desired biological activity of the native protein, that is,retaining herbicide resistance activity. Methods for measuring herbicideresistance activity are well known in the art. See, for example, U.S.Pat. Nos. 4,535,060, and 5,188,642, each of which are hereinincorporated by reference in their entirety.

Bacterial genes, such as the grg and syngrg genes of this invention,quite often possess multiple methionine initiation codons in proximityto the start of the open reading frame. Often, translation initiation atone or more of these start codons will lead to generation of afunctional protein. These start codons can include ATG codons. However,bacteria such as Bacillus sp. also recognize the codon GTG as a startcodon, and proteins that initiate translation at GTG codons contain amethionine at the first amino acid. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may lead to generation of variants of grg and syngrgthat confer herbicide resistance. These herbicide resistance proteinsare encompassed in the present invention and may be used in the methodsof the present invention.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that the DNA sequences of the grg or syngrg genes ofthe invention may be altered by various methods, and that thesealterations may result in DNA sequences encoding proteins with aminoacid sequences different than that encoded by the grg or syngrgsequences disclosed herein. This protein may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions of one or more amino acids of SEQ ID NO:1, 3, 4, 6, 7, 9, 10,12, 13, 15, 17, 18, 20, 21, or 23, including up to about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about15, about 20, about 25, about 30, about 35, about 40, about 45, about50, about 55, about 60, about 65, about 70, about 75, about 80, about85, about 90, about 100 or more amino acid substitutions, deletions orinsertions.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the GRG proteins disclosedherein can be prepared by mutations in the DNA. This may also beaccomplished by one of several forms of mutagenesis and/or in directedevolution. In some aspects, the changes encoded in the amino acidsequence will not substantially affect function of the protein. Suchvariants will possess the desired herbicide resistance activity.However, it is understood that the ability of the GRG proteins disclosedherein to confer herbicide resistance may be improved by one use of suchtechniques upon the compositions of this invention. For example, one mayexpress the grg or syngrg sequences disclosed herein in host cells thatexhibit high rates of base misincorporation during DNA replication, suchas XL-1 Red (Stratagene, La Jolla, Calif.). After propagation in suchstrains, one can isolate the DNA of the invention (for example bypreparing plasmid DNA, or by amplifying by PCR and cloning the resultingPCR fragment into a vector), culture the grg mutations in anon-mutagenic strain, and identify mutated genes with improvedresistance to an herbicide such as glyphosate, for example by growingcells in increasing concentrations of glyphosate and testing for clonesthat confer ability to tolerate increased concentrations of glyphosate.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest, (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art, or, (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent herbicide resistance protein coding regions can be used tocreate a new herbicide resistance protein possessing the desiredproperties. In this manner, libraries of recombinant polynucleotides aregenerated from a population of related sequence polynucleotidescomprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. For example, usingthis approach, sequence motifs encoding a domain of interest may beshuffled between the herbicide resistance gene of the invention andother known herbicide resistance genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedglyphosate resistance activity. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Transformation of Bacterial or Plant Cells

Provided herein are novel isolated genes that confer resistance to anherbicide. Also provided are amino acid sequences of the GRG proteins ofthe invention. The protein resulting from translation of this geneallows cells to function in the presence of concentrations of anherbicide that are otherwise toxic to cells including plant cells andbacterial cells. In one aspect of the invention, the grg or syngrg genesare useful as markers to assess transformation of bacterial or plantcells. Methods for detecting the presence of a transgene in a plant,plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell,propagule, embryo or progeny of the same are well known in the art.

By engineering the genes of the invention to be expressed from apromoter known to stimulate transcription in the organism to be testedand properly translated to generate an intact GRG peptide, and placingthe cells in an otherwise toxic concentration of herbicide, one canidentify cells that have been transformed with the DNA by virtue oftheir resistance to herbicide. By “promoter” is intended a nucleic acidsequence that functions to direct transcription of a downstream codingsequence. The promoter, together with other transcriptional andtranslational regulatory nucleic acid sequences, (also termed as“control sequences”) are necessary for the expression of a DNA sequenceof interest.

Transformation of bacterial cells is accomplished by one of severaltechniques known in the art, including but not limited toelectroporation or chemical transformation (see, for example, Ausubel,ed. (1994) Current Protocols in Molecular Biology, John Wiley and Sons,Inc., Indianapolis, Ind.). Markers conferring resistance to toxicsubstances are useful in identifying transformed cells (having taken upand expressed the test DNA) from non-transformed cells (those notcontaining or not expressing the test DNA). In one aspect of theinvention, the grg or syngrg genes disclosed herein are useful asmarkers to assess transformation of bacterial or plant cells.

Transformation of plant cells can be accomplished in similar fashion. By“plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen). “Transgenic plants” or “transformed plants” or“stably transformed” plants or cells or tissues refer to plants thathave incorporated or integrated exogenous nucleic acid sequences or DNAfragments into the plant cell. By “stable transformation” is intendedthat the nucleotide construct introduced into a plant integrates intothe genome of the plant and is capable of being inherited by progenythereof.

The grg genes of the invention may be modified to obtain or enhanceexpression in plant cells. The herbicide resistance sequences of theinvention may be provided in expression cassettes for expression in theplant of interest. “Plant expression cassette” includes DNA constructs,including recombinant DNA constructs, that are capable of resulting inthe expression of a protein from an open reading frame in a plant cell.The cassette will include in the 5′-3′ direction of transcription, atranscriptional initiation region (i.e., promoter, particularly aheterologous promoter) operably-linked to a DNA sequence of theinvention, and/or a transcriptional and translational termination region(i.e., termination region) functional in plants. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism, such as a selectable marker gene. Alternatively, theadditional gene(s) can be provided on multiple expression cassettes.Such an expression cassette is provided with a plurality of restrictionsites for insertion of the herbicide resistance sequence to be under thetranscriptional regulation of the regulatory regions.

The promoter may be native or analogous, or foreign or heterologous, tothe plant host and/or to the DNA sequence of the invention.Additionally, the promoter may be the natural sequence or alternativelya synthetic sequence. Where the promoter is “native” or “homologous” tothe plant host, it is intended that the promoter is found in the nativeplant into which the promoter is introduced. Where the promoter is“foreign” or “heterologous” to the DNA sequence of the invention, it isintended that the promoter is not the native or naturally occurringpromoter for the operably linked DNA sequence of the invention.“Heterologous” generally refers to the nucleic acid sequences that arenot endogenous to the cell or part of the native genome in which theyare present, and have been added to the cell by infection, transfection,microinjection, electroporation, microprojection, or the like. By“operably linked” is intended a functional linkage between a promoterand a second sequence, wherein the promoter sequence initiates andmediates transcription of the DNA sequence corresponding to the secondsequence. Generally, operably linked means that the nucleic acidsequences being linked are contiguous and, where necessary to join twoprotein coding regions, contiguous and in the same reading frame.

Often, such constructs will also contain 5′ and 3′ untranslated regions.Such constructs may contain a “signal sequence” or “leader sequence” tofacilitate co-translational or post-translational transport of thepeptide of interest to certain intracellular structures such as thechloroplast (or other plastid), endoplasmic reticulum, or Golgiapparatus, or to be secreted. For example, the gene can be engineered tocontain a signal peptide to facilitate transfer of the peptide to theendoplasmic reticulum. By “signal sequence” is intended a sequence thatis known or suspected to result in cotranslational or post-translationalpeptide transport across the cell membrane. In eukaryotes, thistypically involves secretion into the Golgi apparatus, with someresulting glycosylation. By “leader sequence” is intended any sequencethat when translated, results in an amino acid sequence sufficient totrigger co-translational transport of the peptide chain to asub-cellular organelle. Thus, this includes leader sequences targetingtransport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. It may also be preferable to engineer theplant expression cassette to contain an intron, such that mRNAprocessing of the intron is required for expression.

By “3′ untranslated region” is intended a nucleotide sequence locateddownstream of a coding sequence. Polyadenylation signal sequences andother sequences encoding regulatory signals capable of affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNA precursorare 3′ untranslated regions. By “5′ untranslated region” is intended anucleotide sequence located upstream of a coding sequence.

Other upstream or downstream untranslated elements include enhancers.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are well known in the art and include,but are not limited to, the SV40 enhancer region and the 35S enhancerelement.

The termination region may be native with the transcriptional initiationregion, may be native with the herbicide resistance sequence of thepresent invention, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

In one aspect of the invention, synthetic DNA sequences are designed fora given polypeptide, such as the polypeptides of the invention.Expression of the open reading frame of the synthetic DNA sequence in acell results in production of the polypeptide of the invention.Synthetic DNA sequences can be useful to simply remove unwantedrestriction endonuclease sites, to facilitate DNA cloning strategies, toalter or remove any potential codon bias, to alter or improve GCcontent, to remove or alter alternate reading frames, and/or to alter orremove intron/exon splice recognition sites, polyadenylation sites,Shine-Delgarno sequences, unwanted promoter elements and the like thatmay be present in a native DNA sequence. It is also possible thatsynthetic DNA sequences may be utilized to introduce other improvementsto a DNA sequence, such as introduction of an intron sequence, creationof a DNA sequence that in expressed as a protein fusion to organelletargeting sequences, such as chloroplast transit peptides,apoplast/vacuolar targeting peptides, or peptide sequences that resultin retention of the resulting peptide in the endoplasmic reticulum.Synthetic genes can also be synthesized using host cell-preferred codonsfor improved expression, or may be synthesized using codons at ahost-preferred codon usage frequency. See, for example, Campbell andGowri (1990) Plant Physiol. 92:1-11; U.S. Pat. Nos. 6,320,100;6,075,185; 5,380,831; and 5,436,391, U.S. Published Application Nos.20040005600 and 20010003849, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

In one embodiment, the nucleic acids of interest are targeted to thechloroplast for expression. In this manner, where the nucleic acid ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a nucleic acid encoding a transitpeptide to direct the gene product of interest to the chloroplasts. Suchtransit peptides are known in the art. See, for example, Von Heijne etal. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481.

The nucleic acids of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector.” By “transformation vector” is intended aDNA molecule that is necessary for efficient transformation of a cell.Such a molecule may consist of one or more expression cassettes, and maybe organized into more than one “vector” DNA molecule. For example,binary vectors are plant transformation vectors that utilize twonon-contiguous DNA vectors to encode all requisite cis- and trans-actingfunctions for transformation of plant cells (Hellens and Mullineaux(2000) Trends in Plant Science 5:446-451). “Vector” refers to a nucleicacid construct designed for transfer between different host cells.“Expression vector” refers to a vector that has the ability toincorporate, integrate and express heterologous DNA sequences orfragments in a foreign cell.

This plant transformation vector may be comprised of one or more DNAvectors needed for achieving plant transformation. For example, it is acommon practice in the art to utilize plant transformation vectors thatare comprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors.” Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the gene of interest are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science, 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene and in this case“glyphosate”) to recover the transformed plant cells from a group ofuntransformed cell mass. Explants are typically transferred to a freshsupply of the same medium and cultured routinely. Subsequently, thetransformed cells are differentiated into shoots after placing onregeneration medium supplemented with a maximum threshold level ofselecting agent (e.g. “glyphosate”). The shoots are then transferred toa selective rooting medium for recovering rooted shoot or plantlet. Thetransgenic plantlet then grow into mature plant and produce fertileseeds (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida etal. (1996) Nature Biotechnology 14:745-750). Explants are typicallytransferred to a fresh supply of the same medium and cultured routinely.A general description of the techniques and methods for generatingtransgenic plants are found in Ayres and Park (1994) Critical Reviews inPlant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica42:107-120. Since the transformed material contains many cells, bothtransformed and non-transformed cells are present in any piece ofsubjected target callus or tissue or group of cells. The ability to killnon-transformed cells and allow transformed cells to proliferate resultsin transformed plant cultures. Often, the ability to removenon-transformed cells is a limitation to rapid recovery of transformedplant cells and successful generation of transgenic plants. Molecularand biochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest in the genome of transgenicplant.

Generation of transgenic plants may be performed by one of severalmethods, including but not limited to introduction of heterologous DNAby Agrobacterium into plant cells (Agrobacterium-mediatedtransformation), bombardment of plant cells with heterologous foreignDNA adhered to particles, and various other non-particle direct-mediatedmethods (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida etal. (1996) Nature Biotechnology 14:745-750; Ayres and Park (1994)Critical Reviews in Plant Science 13:219-239; Bommineni and Jauhar(1997) Maydica 42:107-120) to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Plants

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

This invention is particularly suitable for any member of the monocotplant family including, but not limited to, maize, rice, barley, oats,wheat, sorghum, rye, sugarcane, pineapple, yams, onion, banana, coconut,and dates.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragments to confirm theintegration of the introduced gene in the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, blotted onto anylon filter according to standard procedures that are routinely used inthe art (Sambrook and Russell, 2001, supra). Expression of RNA encodedby the genes disclosed herein is then tested by hybridizing the filterto a radioactive probe derived from a polynucleotide of the invention,by methods known in the art (Sambrook and Russell, 2001, supra)

Western blot and biochemical assays and the like may be carried out onthe transgenic plants to determine the presence of protein encoded bythe herbicide resistance gene by standard procedures (Sambrook andRussell, 2001, supra) using antibodies that bind to one or more epitopespresent on the herbicide resistance protein.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseintroducing into a plant or plant cell a polynucleotide comprising a grgsequence disclosed herein. As defined herein, the “yield” of the plantrefers to the quality and/or quantity of biomass produced by the plant.By “biomass” is intended any measured plant product. An increase inbiomass production is any improvement in the yield of the measured plantproduct. Increasing plant yield has several commercial applications. Forexample, increasing plant leaf biomass may increase the yield of leafyvegetables for human or animal consumption. Additionally, increasingleaf biomass can be used to increase production of plant-derivedpharmaceutical or industrial products. An increase in yield can compriseany statistically significant increase including, but not limited to, atleast a 1% increase, at least a 3% increase, at least a 5% increase, atleast a 10% increase, at least a 20% increase, at least a 30%, at leasta 50%, at least a 70%, at least a 100% or a greater increase.

In specific methods, the plant is treated with an effectiveconcentration of an herbicide, where the herbicide application resultsin enhanced plant yield. By “effective concentration” is intended theconcentration which allows the increased yield in the plant. Sucheffective concentrations for herbicides of interest are generally knownin the art. The herbicide may be applied either pre- or post emergencein accordance with usual techniques for herbicide application to fieldscomprising crops which have been rendered resistant to the herbicide byheterologous expression of a grg gene of the invention.

Methods for conferring herbicide resistance in a plant or plant part arealso provided. In such methods, a grg polynucleotide disclosed herein isintroduced into the plant, wherein expression of the polynucleotideresults in glyphosate tolerance or resistance. Plants produced via thismethod can be treated with an effective concentration of an herbicideand display an increased tolerance to the herbicide. An “effectiveconcentration” of an herbicide in this application is an amountsufficient to slow or stop the growth of plants or plant parts that arenot naturally resistant or rendered resistant to the herbicide.

In another embodiment, methods for conferring herbicide resistance in aplant or plant part are provided, wherein the plant or plant part isgrown under higher or lower than ambient environmental temperatures asdescribed supra. Glyphosate tolerant EPSP synthase enzymes havingthermal stability at higher or lower temperatures, or have temperatureoptima at higher or lower temperatures, are useful for conferringglyphosate tolerance in plants that are grown under such conditions.

Methods of Controlling Weeds in a Field

Methods for selectively controlling weeds in a field containing a plantare also provided. In one embodiment, the plant seeds or plants areglyphosate resistant as a result of a grg polynucleotide disclosedherein being inserted into the plant seed or plant. In specific methods,the plant is treated with an effective concentration of an herbicide,where the herbicide application results in a selective control of weedsor other untransformed plants. By “effective concentration” is intendedthe concentration which controls the growth or spread of weeds or otheruntransformed plants without significantly affecting theglyphosate-resistant plant or plant seed. Such effective concentrationsfor herbicides of interest are generally known in the art. The herbicidemay be applied either pre- or post emergence in accordance with usualtechniques for herbicide application to fields comprising plants orplant seeds which have been rendered resistant to the herbicide.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Isolation of Glyphosate Resistant EPSP Synthases

Strains capable of growth in presence of glyphosate were isolated byplating samples of soil on HEPES Mineral Salts Medium (HMSM) containingglyphosate as the sole source of phosphorus. Since HMSM contains noaromatic amino acids, a strain must be resistant to glyphosate in orderto grow on this media.

Two grams of soil were suspended in approximately 10 ml of water,vortexed for 15 seconds and permitted to settle for 15 minutes. A 10 μlloopful of this suspension was added to 3 ml of HMSM supplemented with10 mM glyphosate (pH 7.0). HMSM contains (per liter): 10 g glucose, 2 gNH₄SO₄, 9.53 g HEPES, 1.0 ml 0.8 M MgSO₄, 1.0 ml 0.1 M CaCl₂, 1.0 mlTrace Elements Solution (In 100 ml of 1000× solution: 0.1 g FeSO₄.7H₂O,0.5 mg CuSO₄.5H₂O, 1.0 mg H₃BO₃, 1.0 mg MnSO₄.5H₂O, 7.0 mg ZnSO₄.7H₂O,1.0 mg MoO₃, 4.0 g KCl). The culture was grown in a shaker incubator forfour days at 28° C. and then 20 μl was used to inoculate 2.5 ml of freshHMSM containing 10 mM glyphosate as the only phosphorus source. Aftertwo days, 20 μl was used to inoculate another fresh 2.5 ml culture.After 5 days, 20 μl was used to inoculate a fresh 2.5 ml culture. Aftersufficient growth, the culture was plated onto solid media by streakinga 1 μl loop onto the surface of agar plate containing HMSM agarcontaining 100 mM glyphosate as the sole phosphorus source and stored at28° C. The culture was then replated for isolation. The strains listedin Table 1 were among the strains selected due to their ability to growin the presence of high glyphosate concentrations.

TABLE 1 EPSP synthase Strain Name Strain ID Gene Name ATX21561 Unknowngrg33 ATX21563 Unknown grg35 ATX21567 Unknown grg36

Example 2 Isolation of Glyphosate Resistant EPSP Synthases grg37 andgrg39

Strains capable of growth in presence of glyphosate were isolated byplating samples of soil on various growth media containing glyphosate.Some strains were isolated on mineral salts media supplemented withglyphosate. Other strains were isolated under rich media in the presenceof glyphosate and later tested on mineral salts media supplemented withglyphosate. Since the mineral salts media contain no aromatic aminoacids, a strain must be resistant to glyphosate in order to grow on thismedia.

Strains ATX21800 and ATX21804 were isolated by incubation under richconditions and supplemention with glyphosate. These strains were thentested for their ability to grow in the presence of glyphosate withoutaromatic amino acids. Strain ATX21804 was isolated from soil (0.01grams) that was air dried for two days and plated onto nutrient brothagar supplemented with 100 mM glyphosate. A small amount (10 μl) wasthen used to inoculate an eosin methylene blue agar plate containing 300mM glyphosate. ATX21800 was isolated by incubating 0.01 grams soil with3 ml nutrient broth supplemented with 100 mM glyphosate. After initialisolation, each strain was inoculated into Luria Bertani agar plates toconfirm single colony type. These strains were then tested on Brunnerminimal medium containing 100 mM glyphosate and were confirmed to growin the presence of glyphosate without aromatic amino acids.

The strains listed in Table 2 were among the strains selected due totheir ability to grow in the presence of high glyphosate concentrations.

TABLE 2 Strain EPSP synthase Gene Name Strain ID Name ATX21800 Unknowngrg37 ATX21804 Unknown grg39

Example 3 Isolation of Glyphosate Resistant EPSP Synthases grg38 andgrg50

Strains capable of growth in presence of glyphosate were isolated byplating samples of soil on various growth media containing glyphosate.Some strains were isolated on mineral salts media supplemented withglyphosate. Other strains were isolated under rich media in the presenceof glyphosate and later tested on mineral salts media supplemented withglyphosate. Since the mineral salts media contain no aromatic aminoacids, a strain must be resistant to glyphosate in order to grow on thismedia.

Strain ATX20103 was isolated by suspending approximately 2 grams of soilin 10 ml of water, vortexing for 15 seconds and permitting to settle for15 minutes. A 10 μl loopful of this suspension was added to 3 ml of TrisMSM (TMSM) supplemented with 10 mM glyphosate (pH 7.0). TMSM contains(per liter): 10 g glucose, 2 g NH₄SO₄, 12.12 g Tris, 1.0 ml 0.8 M MgSO₄,1.0 ml 0.1 M CaCl₂, 1.0 ml Trace Elements Solution (In 100 ml of 1000×solution: 0.1 g FeSO₄.7H₂O, 0.5 mg CuSO₄.5H₂O, 1.0 mg H₃BO₃, 1.0 mgMnSO₄.5H₂O, 7.0 mg ZnSO₄.7H₂O, 1.0 mg MoO₃, 4.0 g KCl). The culture wasthen incubated at 28° C. for isolation over repeated rounds of selectionand then inoculated onto Luria Bertani agar to confirm single colonytype. ATX20103 was then reconfirmed to grow on TMSM in the presence ofglyphosate without aromatic acids.

Strain ATX21806 was isolated by incubating under rich conditions andsupplementing with glyphosate. This strain was then tested for itsability to growth in the presence of glyphosate without aromatic aminoacids. Strain ATX21806 was isolated from soil (0.01 grams) that had beensuspended in 10 ml water overnight. A small amount (10 μl) was then usedto inoculate an eosin methylene blue agar plate containing 300 mMglyphosate. After each initial isolation, the strain was inoculated intoLuria Bertani agar plates to confirm single colony type. The strain wasthen tested on Brunner minimal medium at 100 mM glyphosate and wasconfirmed to grow in the presence of glyphosate without aromatic aminoacids.

The strains listed in Table 3 were among the strains selected due totheir ability to grow in the presence of high glyphosate concentrations.

TABLE 3 Strain EPSP synthase Gene Name Strain ID Name ATX21806 Unknowngrg38 ATX20103 Rhizobium leguminosarum grg50

Example 4 Cloning of Glyphosate-Resistant EPSP Synthases

Genomic DNA was extracted from the strains described in Tables 1, 2, and3, and the resulting DNA was partially digested with restriction enzymeSau3A 1 to yield DNA fragments approximately 5 kilobases in size. TheseDNA molecules were size selected on agarose gels, purified, and ligatedinto LAMBDA ZAP® vector arms pre-digested with BamH I. The ligated armswere then packaged into phage particles, and phage titers determined asknown in the art. The resulting libraries were amplified by methodsknown in the art to generate a library titer of between 3×10⁷ and 3×10⁸PFU/mL. For each independent library, E. coli (XL1 Blue MRF′) was thenco-transfected with phage from an amplified library as well as M13helper phage to allow mass excision of the library in the form of aninfectious, circular ssDNA as known in the art (Short et al. (1988)Nucleic Acids Research 16:7583-7600). After centrifugation of theco-infected cells, the phage-containing supernatant was heated to 65-70°C. for 15-20 minutes to incapacitate any residual lambda phageparticles. Dilutions of the resulting ssDNA plasmid library weretransfected into a fresh culture of competent E. coli XL-Blue MRF′(aroA) cells (XL1 Blue MRF′). The resulting transfected cells wereplated onto M63 plates containing kanamycin, 0.1 mM IPTG and either 0mM, 20 mM or 50 mM glyphosate.

The E. coli XL-Blue MRF′(aroA) used for the transfection expresses theF-pilus, and also contains a deletion of the aroA gene encoding theendogenous E. coli EPSP synthase enzyme. This strain is also referred toas herein as ΔaroA. This ΔaroA strain is unable to grow on minimal medialacking aromatic amino acids, unless complemented by a functional EPSPsynthase. Since glyphosate is a potent inhibitor of typical,glyphosate-sensitive EPSP synthases, such as type I EPSP synthases,transfected clones expressing a non-glyphosate resistant EPSP synthasewould be able to grown on M63 plates lacking glyphosate, but would beunable to grow on M63 containing either 20 mM or 50 mM glyphosate. Inorder to grow on M63 plates containing 20 mM or 50 mM glyphosate, thecells must contain a plasmid that expresses an EPSP synthase that isboth (1) capable of complementing the ΔaroA mutation of these cells, and(2) resistant to glyphosate. Thus, this screening method allowsidentification of clones containing glyphosate-resistant EPSP synthases.

Colonies growing on 20 mM or 50 mM glyphosate were picked and theirplasmids analyzed by restriction digest to identify plasmids with sharedrestriction patterns. Individual plasmids were sequenced by methodsknown in the art.

Using this approach, as sometimes modified for each library as known andappreciated in the art, library clones containing EPSP synthase geneswere identified for each of the strains listed in Table 4.

Example 5 DNA and Protein Sequences of EPSP Synthases

The DNA sequences of the glyphosate-resistant EPSP synthases wasdetermined for each of the clones described above by methods well knownin the art.

grg33. The DNA sequence of grg33 is provided herein as SEQ ID NO:1. Thepredicted translation product of grg33 (GRG33) is provided herein as SEQID NO:2. A synthetic sequence encoding GRG33 (syngrg33) was alsodesigned and is provided herein as SEQ ID NO:3.

grg35. The DNA sequence of grg35 is provided herein as SEQ ID NO:4. Thepredicted translation product of grg35 (GRG35) is provided herein as SEQID NO:5. A synthetic sequence encoding GRG35 (syngrg35) was alsodesigned and is provided herein as SEQ ID NO:6.

grg36. The DNA sequence of grg36 is provided herein as SEQ ID NO:7. Thepredicted translation product of grg36 (GRG36) is provided herein as SEQID NO:8. A synthetic sequence encoding GRG36 (syngrg36) was alsodesigned and is provided herein as SEQ ID NO:9.

grg37. The DNA sequence of grg37 is provided herein as SEQ ID NO:10. Thepredicted translation product of grg37 (GRG37) is provided herein as SEQID NO:11. A synthetic sequence encoding GRG37 (syngrg37) was alsodesigned and is provided herein as SEQ ID NO:12.

grg38. The DNA sequence of grg38 is provided herein as SEQ ID NO:15. Thepredicted translation product of grg38 (GRG38) is provided herein as SEQID NO:16. A synthetic sequence encoding GRG38 (syngrg38) was alsodesigned and is provided herein as SEQ ID NO:17.

grg39. The DNA sequence of grg39 is provided herein as SEQ ID NO:18. Thepredicted translation product of grg39 (GRG39) is provided herein as SEQID NO:19. A synthetic sequence encoding GRG39 (syngrg39) was alsodesigned and is provided herein as SEQ ID NO:20.

grg50. The DNA sequence of grg50 is provided herein as SEQ ID NO:21. Thepredicted translation product of grg50 (GRG50) is provided herein as SEQID NO:22. A synthetic sequence encoding GRG50 (syngrg50) was alsodesigned and is provided herein as SEQ ID NO:23.

Clones containing each of the grg33, grg35, grg36, grg37, grg38, grg39,and grg50 EPSP synthase genes were deposited at NRRL on Jun. 9, 2006 orJun. 26, 2007 and assigned deposit numbers as in Table 4.

TABLE 4 Clones containing glyphosate-resistant EPSP synthases Strainyielding Original Isolate NRRL EPSPS EPSPS in pBKCMV Number GRG33ATX21561 pAX1947 B-30932 GRG35 ATX21563 pAX1948 B-30933 GRG36 ATX21567pAX1949 B-30934 GRG37 ATX21800 pAX1963 B-30945 GRG38 ATX21806 pAX1964B-30946 GRG39 ATX21804 pAX1965 B-30947 GRG50 ATX20103 pAX1966 B-30948Each of the proteins GRG33, GRG35, and GRG36 showed regions of homologyto EPSP synthase enzymes in the NCBI database by BLAST search. The EPSPSenzyme with the highest protein sequence identity to each GRG enzyme islisted in Table 5.

TABLE 5 Homology of GRG33-GRG36 to known EPSP synthases Strain withhomologous EPSPS Protein enzyme % Identity GRG33 GRG35, S. coelicolor88%, 86% GRG35 GRG33, S. coelicolor 88%, 85% GRG36 Bacillus halodurans53%

The amino acid sequences of GRG33 and GRG35 are 88% identical. A searchof public protein databases with the amino acid sequence of GRG33 showsthat this protein is 86% identical over 430 amino acids to the EPSPsynthase from Streptomyces coelicolor (SEQ ID NO:24 GENBANK® AccessionNo. NP 629359.1), and 82% identical over 430 amino acids to the EPSPsynthase from Streptomyces avermitilis (SEQ ID NO:25; GENBANK® AccessionNo. NP824218.1).

The amino acid sequence of GRG35 similarly is 85% identical over 434amino acids to the EPSP synthase from Streptomyces coelicolor (SEQ IDNO:24; GENBANK®Accession No. NP 629359.1), and 81% identical over 441amino acids to the EPSP synthase from Streptomyces avermitilis (SEQ IDNO:25; GENBANK® Accession No. NP 824218.1).

TABLE 6 Amino acid identity of GRG33 and GRG35 with Streptomyces EPSPsynthases Identity with Identity with EPSP synthase GRG33 GRG35 GRG33 —88% GRG35 88% — Streptomyces coelicolor 84% 82% A3(2) Streptomycesavermitilis MA- 80% 78% 4680 E. coli 30% 29% Maize 30% 29%

A search of public protein databases with the amino acid sequence ofGRG36 shows that this protein is related to the EPSP synthase fromBacillus halodurans, (64% identical over 441 amino acids, SEQ ID NO:26;GENBANK® Accession No. BAB06432.1), and to a lesser extent to the EPSPsynthase from Bacillus clausii (55% identical over 439 amino acids; SEQID NO:27; GENBANK® Accession No. BAD63759.1)

TABLE 7 Amino acid identity of GRG36 with EPSP synthases Identity withEPSP synthase GRG36 Bacillus halodurans 62% Bacillus clausii 54% E. coli31% Maize 34%

A search of public protein databases with the amino acid sequence ofGRG37 shows that this protein is 81% identical to the EPSP synthase fromArthrobacter sp. FB24 (SEQ ID NO:28, GENBANK® Accession No.ZP_(—)00413033.1)

The grg37 open reading frame has two potential start codons. Theupstream ATG (predicted amino acid sequence MTASPMGASADNS (correspondingto amino acid positions 1 through 13 of SEQ ID NO:10)) contains the bestribosome binding site in correct proximity. However, a second downstreamATG may be used. This ORF yields the predicted amino acid sequenceMGASADNS . . . (corresponding to amino acid positions 6 through 13 ofSEQ ID NO:10)). The upstream ATG appears to have a ribosome binding site(“RBS”) that is a better match to the consensus RBS sequence. However,the open reading frame originating from this upstream ATG appears to betranslationally coupled to an upstream open reading frame. Translationalcoupling is one strategy known in the art to be employed by bacteria toensure good initiation and can substitute for a ribosome binding site.The nucleotide sequence for the downstream start site is provided hereinas SEQ ID NO:13, and the encoded amino acid sequence is provided hereinas SEQ ID NO:14.

GRG39 shows 96% amino acid identity to the GRG30 EPSP synthase sequence,and is highly homologous to the GRG29 EPSP synthase sequence describedin U.S. patent application Ser. No. 11/760,570 filed Jun. 8, 2007.

GRG38 shows 94% amino acid identity to the GRG12 EPSP synthase describedin U.S. patent application Ser. No. 11/400,598, filed Apr. 7, 2006(Table 8). GRG38 also contains the domains of the Class III EPSPsynthases described in U.S. patent application Ser. No. 11/400,598.

GRG50 shows 95% amino acid identity to the GRG8 EPSP synthase describedin U.S. patent application Ser. No. 11/315,678 filed Dec. 22, 2005(Table 8). GRG50 also contains the domains of the Class III EPSPsynthases described in U.S. application Ser. No. 11/400,598, filed Apr.7, 2006.

TABLE 8 Comparison with other Class III EPSP synthases Amino acid Aminoacid identity with identity with EPSPS GRG38 GRG50 GRG38 — 65% GRG50 65%— GRG8 65% 95% GRG12 87% 62% GRG6 67% 67% GRG9 64% 70% GRG15 64% 71%GRG5 68% 68% GRG37 67% 68% E. coli (non-Class III) 32% 34% Maize(non-Class III) 32% 31%

Example 6 Cloning of Novel Glyphosate-Resistant EPSP Synthases into anE. coli Expression Vector

The EPSP synthase genes contained in the clones of Table 4 weresub-cloned into the E. coli expression vector pRSF1b (Invitrogen).Resulting clones were confirmed by DNA sequencing, and used to induceexpression of each EPSP synthase in E. coli. The expressed His-taggedprotein was then purified as known in the art.

Example 7 Glyphosate Resistance of GRG33, GRG35, and GRG36 EPSPSynthases

The pRSF1b clones were plated onto M63+ plates containing antibiotic andeither 0 mM or 50 mM glyphosate. Growth was scored after two days growthat 37° C. Each of the three EPSP synthases was observed to conferresistance to 50 mM glyphosate in E. coli cells (Table 9).

TABLE 9 Glyphosate screen Clone in Growth on 50 mM EPSPS pRSF1Bglyphosate Vector — − GRG33 pAX1951 +++ GRG35 pAX1952 +++ GRG36 pAX1953+++

Example 8 Glyphosate Resistance of GRG37 and GRG39 EPSP Synthases

Cells containing the plasmid clones shown in Table 4 were plated ontoM63+ plates containing antibiotic and either 0 mM or 20 mM glyphosate.Growth was scored after two days growth at 37° C. Each of the EPSPsynthases was observed to confer resistance to 20 mM glyphosate in E.coli cells (Table 10).

TABLE 10 Glyphosate screen Growth on 20 mM EPSPS Plasmid Cloneglyphosate Vector — − GRG37 pAX1963 ++ GRG39 pAX1965 ++

Example 9 Glyphosate Resistance of GRG38 and GRG50 EPSP Synthases

Cells containing the plasmid clones shown in Table 4 were plated ontoM63+ plates containing antibiotic and either 0 mM or 20 mM glyphosate.Growth was scored after two days growth at 37° C. Each of the EPSPsynthases was observed to confer resistance to 20 mM glyphosate in E.coli cells (Table 11).

TABLE 11 Glyphosate screen Growth on 20 mM EPSPS Plasmid Cloneglyphosate Vector — − GRG38 pAX1964 ++ GRG50 pAX1966 ++

Example 10 Engineering grg33, grg35, grg36, grg37, grg38, grg39, grg50,syngrg33, syngrg35, syngrg36, syngrg37, syngrg38, syngrg39, and syngrg50for Plant Transformation

The open reading frame (ORF) for each of the grg genes is amplified byPCR from a full-length cDNA template. Hind III restriction sites areadded to each end of the or F during PCR. Additionally, the nucleotidesequence ACC is added immediately 5′ to the start codon of the gene toincrease translational efficiency (Kozak (1987) Nucleic Acids Research15:8125-8148; Joshi (1987) Nucleic Acids Research 15:6643-6653). The PCRproduct is cloned and sequenced, using techniques well known in the art,to ensure that no mutations are introduced during PCR. The plasmidcontaining the grg PCR product is digested with, for example, Hind IIIand the fragment containing the intact or F is isolated.

One may generate similar constructs that contain a chloroplast targetingsequence linked to the polynucleotide of the invention by methods knownin the art.

A DNA fragment containing the EPSP synthase (and either containing ornot containing a chloroplast targeting sequence) is cloned into aplasmid, for example at the Hind III site of pAX200. pAX200 is a plantexpression vector containing the rice actin promoter (McElroy et al.(1991) Molec. Gen. Genet. 231:150-160), and the PinII terminator (An etal. (1989) The Plant Cell 1:115-122). The promoter—gene—terminatorfragment (or the promoter-leader-gene-terminator fragment) from thisintermediate plasmid is subcloned into a plasmid such as pSB11 (JapanTobacco, Inc.) to form a final plasmid, referred to herein as, forexample, pSB11GRG33. pSB11GRG33 is organized such that the DNA fragmentcontaining, for example, the promoter—grg36—terminator construct (or thepromoter-leader-grg36—terminator construct) may be excised byappropriate restriction enzymes and also used for transformation intoplants, for example, by aerosol beam injection. The structure ofpSB11GRG33 is verified by restriction digest and gel electrophoresis, aswell as by sequencing across the various cloning junctions. The samemethods can be used to generate a final plasmid for each of the grggenes described herein.

The plasmid is mobilized into Agrobacterium tumefaciens strain LBA4404which also harbors the plasmid pSB1 (Japan Tobacco, Inc.), usingtriparental mating procedures well known in the art, and plating onmedia containing antibiotic. Plasmid pSB11GRG36 carries spectinomycinresistance but is a narrow host range plasmid and cannot replicate inAgrobacterium. Antibiotic resistant colonies arise when pSB11GRG36integrates into the broad host range plasmid pSB1 through homologousrecombination. The resulting cointegrate product is verified by Southernhybridization. The Agrobacterium strain harboring the cointegrate can beused to transform maize, for example, by the PureIntro method (JapanTobacco).

Example 11 Transformation grg33, grg35, grg36, grg37, grg38, grg39,grg50, syngrg33, syngrg35, syngrg36, syngrg37, syngrg38, syngrg39, andsyngrg50 into Plant Cells

Maize ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, such as DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of 1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine;100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D). However, media and saltsother than DN62A5S are suitable and are known in the art. Embryos areincubated overnight at 25° C. in the dark. However, it is not necessaryper se to incubate the embryos overnight.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for about 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to express the GRG proteins of the presentinvention in plant cells are accelerated into plant tissue using anaerosol beam accelerator, using conditions essentially as described inPCT Publication No. WO/0138514. After beaming, embryos are incubated forabout 30 min on osmotic media, and placed onto incubation mediaovernight at 25° C. in the dark. To avoid unduly damaging beamedexplants, they are incubated for at least 24 hours prior to transfer torecovery media. Embryos are then spread onto recovery period media, forabout 5 days, 25° C. in the dark, then transferred to a selection media.Explants are incubated in selection media for up to eight weeks,depending on the nature and characteristics of the particular selectionutilized. After the selection period, the resulting callus istransferred to embryo maturation media, until the formation of maturesomatic embryos is observed. The resulting mature somatic embryos arethen placed under low light, and the process of regeneration isinitiated by methods known in the art. The resulting shoots are allowedto root on rooting media, and the resulting plants are transferred tonursery pots and propagated as transgenic plants.

Materials

TABLE 12 DN62A5S Media Components Per Liter Source Chu's N6 Basal 3.98g/L Phytotechnology Salt Mixture Labs (Prod. No. C 416) Chu's N6 1 mL/L(of 1000x Stock) Phytotechnology Vitamin Solution Labs (Prod. No. C 149)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline 1.4 g/L Phytotechnology Labs Casamino acids 100 mg/L FisherScientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1 mL/L(of 1 mg/mL Stock) Sigma D-7299)

The pH of the solution is adjusted to pH 5.8 with 1N KOH/1N KCl, Gelrite(Sigma) is added at a concentration up to 3 g/L, and the media isautoclaved. After cooling to 50° C., 2 ml/L of a 5 mg/ml stock solutionof silver nitrate (Phytotechnology Labs) is added.

Example 12 Transformation of grg33, grg35, grg36, grg37, grg38, grg39,grg50, syngrg33, syngrg35, syngrg36, syngrg37, syngrg38, syngrg39, andsyngrg50 into Maize Plant Cells by Agrobacterium-Mediated Transformation

Ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, and incubated overnight at 25°C. in the dark. However, it is not necessary per se to incubate theembryos overnight. Embryos are contacted with an Agrobacterium straincontaining the appropriate vectors for Ti plasmid mediated transfer forabout 5-10 min, and then plated onto co-cultivation media for about 3days (25° C. in the dark). After co-cultivation, explants aretransferred to recovery period media for about five days (at 25° C. inthe dark). Explants are incubated in selection media for up to eightweeks, depending on the nature and characteristics of the particularselection utilized. After the selection period, the resulting callus istransferred to embryo maturation media, until the formation of maturesomatic embryos is observed. The resulting mature somatic embryos arethen placed under low light, and the process of regeneration isinitiated as known in the art. The resulting shoots are allowed to rooton rooting media, and the resulting plants are transferred to nurserypots and propagated as transgenic plants.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising the amino acid sequence of SEQ ID NO:8; b) apolypeptide encoded by the nucleotide sequence of SEQ ID NO:7 or 9; c) apolypeptide comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:8, whereinsaid polypeptide has herbicide resistance activity; d) a polypeptidethat is encoded by the herbicide resistance nucleotide sequence of theDNA insert of the plasmid deposited as Accession No. NRRL B-30934. 2.The polypeptide of claim 11 further comprising a heterologous amino acidsequence.